Name: highly multiplexed, super-resolution imaging of t cells using madstorm
Uploaded: 2017-06-24T12:30:00
Description: Watch this Scientific Journal Video about Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM at JoVE.com

Nous remercions Xufeng Wu d&#39;avoir accès au microscope STORM. Cette recherche a été appuyée par le Programme de recherche intra-muros du Centre national de recherche sur le cancer (NCI) et l&#39;Institut national du cancer du poumon et du sang (NHLBI).

Attention: veuillez consulter toutes les fiches signalétiques pertinentes (fiche signalétique) avant utilisation. Plusieurs des produits chimiques utilisés dans ce protocole sont toxiques et cancérogènes. Utilisez toutes les pratiques de sécurité appropriées lors de l&#39;exécution du protocole, y compris l&#39;utilisation de commandes techniques (hotte aspirante, boîte à gants) et équipement de protection individuelle (lunettes de sécurité, gants, blouse de laboratoire, pantalons complets, chaussures ferm...

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Les procédures de multiplexage séquentiel, de correction de dérive et d&#39;alignement dans madSTORM permettent une visualisation précise et hautement multiplexée des structures hétérogènes dans les cellules. 10 En outre, madSTORM évite les limitations de la STORM multicolore, telles que l&#39;aberration chromatique et les propriétés de décodage / émission de photos non optimales des colorants non lointains 9 , 12 . Comme la ...

Une variété de techniques de microscopie de super résolution ont été développées pour surmonter la limite de diffraction du microscope optique (~ 200 nm). Parmi ceux-ci se trouve une catégorie de techniques appelées microscopie de localisation de molécules simples (SMLM) qui comprend la microscopie de localisation photo-activation (PALM) et la microscopie de reconstruction optique stochastique (STORM). Les techniques SMLM participent à l&#39;utilisation de fluorophores qui peuvent être commutés entre des états fluorescents et des états décalés (numérisés ou numérisés), ce qui permet une localisation séquentielle de la fluorescence à partir de molécules 1 , 2 , 3 . En raison de sa compatibilité avec les colorants et les microscopes disponibles dans le commerce, STORM direct (dSTORM) est devenu une technique SMLM largement adoptée 4 . DSTORM peut atteindre systématiquement une précision de localisation de ~ 10 nm, définie comme l&#39;incertitude dans le calcul du centre d&#39;un diffractFonction de propagation des points à limitation d&#39;ions (PSF). Cependant, malgré la précision élevée estimée à l&#39;aide des algorithmes de localisation 5 , 6 , 7 , une détermination précise de l&#39;emplacement réel des molécules individuelles a été entravée par un certain nombre de problèmes. Tout d&#39;abord, le mouvement mécanique du stade du microscope pendant l&#39;acquisition de l&#39;image ajoute une grande incertitude quant à la précision de localisation. Comme les images SMLM sont obtenues sur des milliers de trames temporelles, les mouvements en nano-échelle du stade du microscope peuvent compromettre de manière significative la précision de l&#39;image finale de super résolution 8 . Pour compenser le mouvement de l&#39;étape pendant l&#39;acquisition de l&#39;image, la dérive de l&#39;étage est généralement estimée à partir d&#39;un ajustement basé sur la régression des localisations binées de l&#39;image elle-même (corrélation croisée) ou des localisations séquentielles des marqueurs fiduciaires (correction fiduciaire) 1 , 9 . HowevCes méthodes nécessitent l&#39;optimisation de multiples paramètres pour chaque pile d&#39;images et ne peuvent pas tenir compte des mouvements de l&#39;étage à des échelles de temps courtes telles que des vibrations mécaniques. Les nanoparticules d&#39;or et les perles fluorescentes multicolores ont été utilisés comme marqueurs fiduciaires dans le SMLM, mais ils ne sont pas stables à la photo et entraînent une précision nettement inférieure après la correction de la dérive que les nano-diamants fluorescents au centre d&#39;inoccupation d&#39;azote (FND) utilisés In madST 10 . En plus de la limite de diffraction, le microscope optique est encore limité par des limites spectrales. La visualisation simultanée de multiples cibles nécessite des sondes fluorescentes avec des profils spectraux qui ne se chevauchent pas, limitant généralement le microscope optique à fluorescence à 6 couleurs et le SMLM à 2-3 couleurs 4 , 11 , 12 . En outre, l&#39;aberration chromatique non linéaire provoque un désalignement des images multicolores, wQui nécessitent des procédures d&#39;alignement étendues utilisant des marqueurs fiduciaires multicolores 8 , 13 . Pour surmonter ces limites, des études antérieures ont imaginé de multiples cibles en utilisant un photoblanchissement répétitif ou une trempe chimique des fluorophores liés aux séquences 14 , 15 , 16 , 17 , 18 , 19 . Bien que ces méthodes puissent surmonter les limites spectrales de la microscopie, le blanchiment par fluorescence est connu pour être un processus toxique 20 , et le blanchiment prolongé ou la trempe peut provoquer des effets secondaires indésirables tels que la perte de réticulation. En outre, l&#39;accumulation de sondes fluorescentes pourrait conduire à un blocage stérique des sites de liaison dans l&#39;échantillon, empêchant le multiplexage à grande échelle et le ciblage robuste des epitopes. Pour éviter de telles interférences stériques, un récent sLe multiplexage a eu lieu à l&#39;aide d&#39;un échange stochastique de fragments de protéines diffusant librement 21 . Alors que cette méthode permet un étiquetage dense des structures cellulaires, il nécessite une préparation biochimique étendue pour isoler les fragments de peptides, ne peut pas localiser des positions de molécules simples et ne facilite pas facilement le multiplexage à grande échelle en utilisant des sondes disponibles dans le commerce. Nous présentons un protocole vidéo détaillé décrivant la liaison séquentielle et l&#39;élution d&#39;anticorps fluorescents pour l&#39;imagerie multiplexée, la technologie dSTORM (MadSTORM) limitée par l&#39;anticorps et l&#39;utilisation de nano-diamants fluorescents pour obtenir une correction et un alignement précis de la dérive.

<table border="0" cellpadding="0" cellspacing="0" width="598">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">8 well coverslip chamber</td>
			<td style="width:117px;">Lab-tek</td>
			<td style="width:124px;">155409</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.1% Poly-L-Lysine solution</td>
			<td>Sigma-Aldrich</td>
			<td>P8920</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NV-100nm Fluorescent Nano-diamond</td>
			<td>Adamas</td>
			<td>Red FND</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-CD3&#949; antibody</td>
			<td>BD Biosciences</td>
			<td>555329</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin, Fraction V</td>
			<td>KSE Scientific</td>
			<td>98-100P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton-X</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10% Paraformaldehyde</td>
			<td>EMS</td>
			<td>15712</td>
			<td>Carcinogen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin from fresh water fish skin</td>
			<td>Sigma-Aldrich</td>
			<td>G7041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa 647 antibody labeling kit</td>
			<td>Thermo Fisher</td>
			<td>A20186</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Chloride hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>138924</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PIPES</td>
			<td>Sigma-Aldrich</td>
			<td>P6757</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M7154</td>
			<td>Highly toxic, air sensitive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cysteamine</td>
			<td>Sigma-Aldrich</td>
			<td>30070</td>
			<td>Highly toxic&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cyclooctatetraene 98%</td>
			<td>Sigma-Aldrich</td>
			<td>138924</td>
			<td>Highly toxic, air sensitive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x PBS</td>
			<td>KD Medical</td>
			<td>RGF-3210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x TBS</td>
			<td>KD Medical</td>
			<td>RGF-3385</td>
			<td></td>
		</tr>
	</tbody>
</table>

highly multiplexed, super-resolution imaging of t cells using madstorm

L&#39;imagerie de structures cellulaires hétérogènes utilisant une microscopie de localisation à une seule molécule a été entravée par une précision de localisation insuffisante et une capacité de multiplexage. À l&#39;aide de marqueurs fluorescents de nano-diamant fiducial, nous décrivons les procédures de correction de dérive et d&#39;alignement requises pour obtenir une grande précision dans un microscope à localisation à une seule molécule. En outre, une nouvelle stratégie de multiplexage, madSTORM, est décrite dans laquelle de multiples molécules sont ciblées dans la même cellule en utilisant la liaison séquentielle et l&#39;élution d&#39;anticorps fluorescents. MadSTORM est démontré sur une cellule T activée pour visualiser les emplacements de différents composants dans une structure multi-protéine liée à la membrane appelée micro-cluster de récepteur de cellules T. En outre, l&#39;application de madSTORM comme outil général pour la visualisation des structures multi-protéines est discutée.

La méthode séquentielle d&#39;élution et de coloration a été utilisée pour produire l&#39;image madSTORM multiplexée de microclusters et d&#39;autres structures dans une cellule T Jurkat activée ( Figure 1 , voir les alignements des figures). Chaque image pseudo-colorée représente une série d&#39;imagerie madSTIME acquise aux étapes 1.1.1 à 1.3.13. Comme décrit précédemment 10 , le champ de vision doit être surveillé...

Watch this Scientific Journal Video about Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM at JoVE.com

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Nous démontrons une méthode pour imaginer des molécules multiples dans des nano-structures hétérogènes à une seule précision de la molécule en utilisant la liaison séquentielle et l&#39;élution d&#39;anticorps marqués par fluorescence.

Imagerie haute résolution et ultra-résolution des cellules T à l&#39;aide de madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

The overall goal of this imaging protocol is to visualize multiple proteins in an activated T cell, using single molecule localisation microscopy. This method can help answer key questions in the T cell activation field such as, the single molecule distribution of TC or microcluster components. The main advantage of this technique is that, it allows greater multiplexing than achieved by current supervised solution microscopy methods.To begin, directly conjugate antibodies with Alexa 647 dye, using the Alexa 647 antibody labeling kit, following the labeling protocol provided by the manufacturer. Centrifuge the labeled antibodies for five minutes at 20, 800 times G.Following centrifugation, collect the supernatant to remove the aggregated antibodies. Next, coat A12 cover slip chambers with 250 microliters of 01%poly-l-lyzine for 15 minutes.Then, aspirate the solution without rinsing and dry the chambers at 65 degree celsius for 30 minutes. Prepare dilution of 100 nanometer fluorescent nanodiamonds or FNDs, in one PBS, test various dilutions to ensure that enough FNDs will be visible in each field. Vortex the diluted FNDs for one minute.Next, sonicate the FND supernatant for 30 seconds at a high power setting. Incubate the sonicated FND supernatant in a poly-l-lyzine coated A12 cover slip chamber for 30 minutes at room temperature. After washing the chamber five times, with 1x PBS, visualize the FND coated chamber using 647 nanometer laser excitation on the turf microscope.Use a 100x objective tealed a 61 by 61 micron field of view. Ideally, four to 10 individual FNDs should be visible in the field of view, given the appropriate dilution of FNDs, with at least one FND present in each quadrant of the imaging field. Once the proper density of FNDs has been achieved, add 250 microliters of anti-CD3 antibody to each well.Incubate the chamber for one hour at 37 degree celsius or overnight at four degree celsius. Following incubation, remove the solution from the wells and add 1x PBS for 30 seconds. Repeat this wash step five times.Spin one milliliter of Jurkat T cells at 800 times G for six minutes. Jurkat T cells should ideally be at a concentration of 0.5 to one million cells per milliliter concentration before spinning. Re-suspend the cells in 300 microliters of 1x HBS solution.Meanwhile, add 150 microliters of 1x HBS solution to each well of the chamber and incubate at 37 degree celsius for 15 minutes. Then, add 50 microliters of the re-suspended Jurkat T cells to each well and incubate for three minutes at 37 degree celsius. Next, add 300 microliters of 4%paraformaldehyde to each well and incubate for 30 minutes at 37 degree celsius.After washing three times with 1x PBS, permeabilise the cells, by adding 250 microliters of 01%Triton x solution for five minutes at room temperature. Following three additional washes with 1x PBS, add 250 microliters of 1%fish gelatin solution to each well for 30 minutes at room temperature. Finally, wash the cells three more times with 1x PBS.Add 200 microliters of labeled antibody at 0.1 to 0.5 micrograms per milliliter to the fixed cells for one hour at room temperature. After washing the cells five times with 1x PBS, add one milliliter of STORM buffer and cover the chamber with a glass cover slip to limit exposure. Use clamps on the microscope stage to hold the cover slip chamber in position.This step is critical for large scale multiplexing, since the same field of view must be present after several rounds of washing and incubation. Using a low 647 nanometer laser power in turf mode, locate a stained cell with at least three FNDs in the field of view. Increase the 647 nanometer laser power and acquire the images.Typical parameters can be found in the text protocol. Wash the cells five times with 1x tris-buffered saline or 1x TBS. Then, add one milliliter of elution buffer and incubate at room temperature for one minute.Repeat the elution three times. Use the exact elution conditions and a number of elution rinses needed to remove the signal as specified for each antibody. Then, wash the cells three times with 1x TBS and add one milliliter of 1x PBS.Proper execution of this elution step, will result in removal of previously bound antibody and allow higher efficiency for subsequent antibody labeling. It is important to use TBS for washes to minimize precipitation between elution steps. Photo bleach the cells using a 647 nanometer laser at high power, with a 405 nanometer laser at two to five milliwatts to photo activate Alexa 647 dyes in the dark state.Wait until all remaining signal from non-eluted antibody is photo bleached. Typically, this takes two to five seconds of laser exposure. Acquire sample images, using the STORM settings after illusion and photo bleaching, to confirm removal of the signal.Next, add 250 microliters of 4%paraformaldehyde for 15 minutes. This prevents reverse cross-linking of fixed molecules in the cell. Wash the cells three times with 1x PBS.Repeat these steps for sequential labeling of multiple targets. Finally, perform drift correction and alignment of multiplex image stacks at described in the text protocol. The sequential illusion and standing method was used to produce the multiplex MET STORM image of microclusters and other structures in an activated Jurkat T cell.The final MET STORM image has been corrected for a drift and alignment, using average fiducial correction and FND fiducial markers. Shown here, are single molecule localisation microscopy images of a single FND localized in 30, 000 image frames before and after drift correction with average fiducial correction. Similarly, here are single molecule localisation microscopy images of an activated Jurkat T cell stained with anti-phosphorelated SLP76 antibody and FND futusial markers, before and after drift correction with average futusial correction.After watching this video, you should have a good understanding of how to perform multiplexed super resolution imaging of proteins in an activated T cell, using MET STORM. Once mastered, this technique can be done in three hours for each multiplexing round. While attempting this procedure, it's important to remember to keep the specimen in the same position on the microscope stage.This can be done using stage clamps. Don't forget that working with our modified STORM buffer, can be extremely hazardous and precautions such as, respirators and goggles should always be taken while performing this procedure.

Research

JoVE Journal

Immunology and Infection

Retroviral Transduction of T-cell Receptors in Mouse T-cells

Transduction rétrovirale des récepteurs de cellules T dans les lymphocytes T de souris

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Recherche

Immunologie et infection

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Ex vivo l&#39;imagerie de cellules T dans murin tranches avec des ganglions lymphatiques et la microscopie confocale Widefield

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

Expansion du sang périphérique humain cellules T γδ utilisant zolédronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

Génération de cellules T régulatrices induites de l&#39;homme primaires cellules T naïves et mémoire

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Très Résolu microscopie intravitale Striped-éclairage de centres germinaux

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Moyenne de la glycoprotéine d&#39;enveloppe virale Pointes de Electron Cryotomography reconstructions utilisant Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

Super-résolution de l&#39;imagerie cellulaire Synapse immunologique Natural Killer sur un verre soutenu par Planar lipidique en deux couches

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Analyse de<em&gt; Yersinia enterocolitica</em&gt; Effecteur translocation dans les cellules hôtes à l&#39;aide de bêta-lactamase effecteur Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Actine corticale Débit en cellules T quantifiée par spatio-temporelle image spectroscopie par corrélation de Structured Illumination Microscopy données

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

In Vitro Differentiation of Human CD4 + FOXP3 cellules + Induced T régulatrices (de iTregs) de Naïf cellules T CD4 + en utilisant un protocole TGF-β contenant